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PubMed1995

[Spatial structure of bacterioopsin transmembrane segments C, E, and G from two-dimensional 1H-NMR data].

The spatial structure of synthetic peptides with transmembrane segment sequences C (residues 67-106), E (128-162), and G (190-233) of bacterioopsin from Halobacterium halobium solubilized in methanol-chloroform 1:1 containing 0.1 M LiClO4 was computed based on 2D 1H NMR data. Segment C forms a right alpha-helix between Pro77 and Val101. The residue Pro91 within the alpha-helix induces a 25 degrees "kink". Segment E forms a right alpha-helix between Val136 and Ser158. Segment G includes alpha-helical region, which begins from CO of Ile198 and ends at N alpha-H of Arg227. The torsion angles, chi 1, of the side chains were unambiguously determined for most of the residues within the alpha-helical regions. Conformations of some side chains of the transmembrane segment residues in the solution were found to differ from their previously reported conformations determined by electron microscopy. Conformations of the sites terminating the C and E segment alpha-helices were refined.

Amino Acid Sequence↗

PubMed2004

iMOT: an interactive package for the selection of spatially interacting motifs.

Functional selection and three-dimensional structural constraints of proteins relate to the retention of significant sequence similarity between proteins of similar fold and function despite poor overall sequence identity and evolutionary pressures. We report the availability of 'iMOT' (interacting MOTif) server, an interactive package for the automatic identification of spatially interacting motifs among distantly related proteins sharing similar folds and possessing common ancestral lineage. Spatial interactions between conserved stretches of a protein are evaluated by calculations of pseudo-potentials that describe the strength of interactions. Such an evaluation permits the automatic identification of highly interacting conserved regions of a protein. Interacting motifs have been shown to be useful in searching for distant homologues and establishing remote homologies among the largely unassigned sequences in genome databases. Information on such motifs should also be of value in protein folding, modelling and engineering experiments. The iMOT server can be accessed from http://www.ncbs.res.in/~faculty/mini/imot/iMOTserver.html. Supplementary Material can be accessed from: http://www.ncbs.res.in/~faculty/mini/imot/supplementary.html.

Amino Acid Motifs↗

PubMed2000

Patterns of regional brain activation associated with different forms of motor learning.

To examine the variations in regional cerebral blood flow during execution and learning of reaching movements, we employed a family of kinematically and dynamically controlled motor tasks in which cognitive, mnemonic and executive features of performance were differentiated and characterized quantitatively. During 15O-labeled water positron emission tomography (PET) scans, twelve right-handed subjects moved their dominant hand on a digitizing tablet from a central location to equidistant targets displayed with a cursor on a computer screen in synchrony with a tone. In the preceding week, all subjects practiced three motor tasks: 1) movements to a predictable sequence of targets; 2) learning of new visuomotor transformations in which screen cursor motion was rotated by 30 degrees -60 degrees; 3) learning new target sequences by trial and error, by using previously acquired routines in a task placing heavy load on spatial working memory. The control condition was observing screen and audio displays. Subtraction images were analyzed with Statistical Parametric Mapping to identify significant brain activation foci. Execution of predictable sequences was characterized by a modest decrease in movement time and spatial error. The underlying pattern of activation involved primary motor and sensory areas, cerebellum, basal ganglia. Adaptation to a rotated reference frame, a form of procedural learning, was associated with decrease in the imposed directional bias. This task was associated with activation in the right posterior parietal cortex. New sequences were learned explicitly. Significant activation was found in dorsolateral prefrontal and anterior cingulate cortices. In this study, we have introduced a series of flexible motor tasks with similar kinematic characteristics and different spatial attributes. These tasks can be used to assess specific aspects of motor learning with imaging in health and disease.

Adult↗

PubMed2001

Embedded MR fluoroscopy: high temporal resolution real-time imaging during high spatial resolution 3D MRA acquisition.

A method termed "embedded fluoroscopy" for simultaneously acquiring a real-time sequence of 2D images during acquisition of a 3D image is presented. The 2D images are formed by periodically sampling the central phase encodes of the slab-select direction during the 3D acquisition. The tradeoffs in spatial and temporal resolution are quantified by two parameters: the "redundancy" (R), the fraction of the 3D acquisition sampled more than once; and the "effective temporal resolution" (T), the time between temporal updates of the central views. The method is applied to contrast-enhanced MR angiography (CE-MRA). The contrast bolus dynamics are portrayed in real time in the 2D image sequence while a high-resolution 3D image is being acquired. The capability of the 2D acquisition to measure contrast enhancement with only a 5% degradation of the spatial resolution of the 3D CE-MR angiogram is shown theoretically. The method is tested clinically in 15 CE-MRA patient studies of the carotid and renal arteries.

Feasibility Studies↗

PubMed1983

Secondary structure of the lac repressor DNA-binding domain by two-dimensional 1H nuclear magnetic resonance in solution.

A recently proposed approach for spatial structure determination in noncrystalline proteins by nuclear magnetic resonance was applied to the lac repressor DNA-binding domain. On the basis of sequence-specific 1H NMR assignments, the location of alpha-helices in the amino acid sequence was determined from nuclear Overhauser enhancement data and from amide proton exchange studies. These investigations provide detailed experimental data on the structure of a noncrystalline DNA-binding protein. The results support the hypothesis advanced by others that sequence-specific interactions between lac repressor and DNA are mediated by a particular spatial arrangement of two alpha-helices common to various different DNA-binding proteins.

Amino Acid Sequence↗

PubMed1999

Evaluation of the inner ear by 3D fast asymmetric spin echo (FASE) MR imaging: phantom and volunteer studies.

The 3D fast asymmetric spin echo (FASE) method combines the half-Fourier technique and 3D fast spin echo (FSE) sequence. The advantage of this method is that it maintains the same spatial resolution as FSE while markedly reducing the imaging time. The purpose of the present study was to evaluate the usefulness of the 3D FASE technique in displaying the inner ear structure using phantom and volunteer studies. 3D FSE sequence images were obtained for comparison, and the optimum 3D FASE sequence was investigated on a 1.5T MR scanner. The results of phantom experiments showed increased signal-to-noise ratio (SNR) with prolonging repetition time (TR) on both 3D FASE and 3D FSE sequences. Although the SNR of 3D FASE images was 20-25% lower than that of 3D FSE images with the same TR, the SNR per minute with 3D FASE was about twice that with 3D FSE. On 3D FASE images, a higher spatial resolution was obtained with 2- or 4-shot images than with single-shot images. However, no significant difference was observed between 2-shot and 4-shot images. In the volunteer study, 3D FASE images using a TR of 5000 ms and an effective echo time (TEeff) of 250 ms showed a high SNR and spatial resolution and provided excellent contrast between cerebrospinal fluid and nerves in the internal auditory canal. The highest contrast was achieved in the 2-shot/2 number of excitations sequence. 3D FASE provides the same image quality as 3D FSE with a significant reducing in imaging time, and gives strong T2-weighted images. This method enables detailed visualization of the tiny structures of the inner ear.

Adult↗

PubMed1989

Photoaffinity labeling of terminal deoxynucleotidyl transferase. 2. Identification of peptides in the nucleotide binding domain.

Terminal deoxynucleotidyl transferase (terminal transferase) was specifically modified in the nucleotide binding site by the substrate photoaffinity analogue [gamma-32P]-8-azido-dATP. The alpha and beta polypeptides of photolabeled terminal transferase were resolved by high-performance liquid chromatography. The beta polypeptide was digested with trypsin and fractionated by reverse-phase chromatography. Two 32P-containing fractions were isolated and subjected to amino acid sequence analysis. Peptides were identified as Ile209-Lys232 (B26) and Val233-Lys239 (B27). Peptide B26 was further resolved into two overlapping species; one contained an additional lysine residue at the N-terminus which resulted from tryptic cleavage between Lys207 and Lys208. In order to ensure that the sequenced peptides corresponded to the photolabeled species, we devised an anion-exchange procedure to isolate photolabeled peptides from the mixture. Analysis of photolabeled peptides from terminal transferase alpha beta using DEAE-cellulose chromatography followed by reverse-phase HPLC confirmed that the photolabeled species were peptides B26 and B27. Peptide B26, the major photolabeled species, contained a conserved octapeptide region found in several eucaryotic DNA polymerases. In addition, peptide B27 was flanked by a sequence that has been implicated in triphosphate binding in other proteins. Structure predictions, based on sequence data, place the two peptides identified by photolabeling in spatial proximity consistent with the participation of both in the nucleotide binding domain.

Adenosine Triphosphate↗

PubMed1998

Regulation of the Caenorhabditis elegans gut cysteine protease gene cpr-1: requirement for GATA motifs.

Expression of the Caenorhabditis elegans cysteine protease gene cpr-1 is regulated both spatially and temporally. In situ hybridisation and Northern blot analysis have shown that this gene is expressed exclusively in gut cells of all developmental stages except the embryo. We now show by transgenic transformation with cpr-1/lac Z reporter gene constructs that a sequence contained within the cpr-1 5' flanking region can direct this spatial and temporal expression. Deletion analysis of the cpr-1 promoter indicates that as little as 212 bp of upstream sequence is sufficient for this expression, although more upstream sequence may be involved in quantitative regulation of expression. Mutation of two GATA-like sequence elements at positions -51 and -147 upstream of the transcription start site ablates all expression, indicating an essential role in cpr-1 regulation. A concatemer of the cpr-1 -147 GATA motif placed upstream of minimal promoter/lac Z reporter gene constructs results in strong reporter gene expression in gut cells of larval stages and also in embryos. Weak expression is also detected in hypodermal cells. This pattern is reversed in the adult stage with strong expression in hypodermal cells and weaker expression in gut cells. Our findings suggest that spatial and temporal regulation of the cpr-1 gene is complex and involves activation by a GATA-like transcription factor.

Animals↗

PubMed1997

Magnetic resonance renography: optimisation of pulse sequence parameters and Gd-DTPA dose, and comparison with radionuclide renography.

The aim of this study was to assess the feasibility of magnetic resonance renography (MRR) using gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) in comparison with conventional radionuclide renography (RR) using technetium-99m-DTPA (99mTc-DTPA). MRR has many advantages over RR, including lack of ionising radiation, increased spatial resolution, and visible background anatomy. By optimising the pulse sequence, we developed an MRR protocol in which signal intensity is linear with Gd-DTPA concentration over a clinically relevant range. Twenty-nine patients and a volunteer were studied using this protocol. Magnetic resonance renography was performed using three different doses of Gd-DTPA: 0.1 mmol kg-1 (n = 13), 0.05 mmol kg-1 (n = 7), and 0.025 mmol kg-1 (n = 9). Each patient was also assessed using radionuclide renography. The resulting renograms were assessed in terms of time to peak signal intensity, signal decrease after peak, and kidney function ratios calculated from both the areas underneath and the slopes of the uptake curves. We have shown that the MR renograms obtained using low dose Gd-DTPA correlate best with the radionuclide renograms. Remaining discrepancies may be explained by variations in the injection procedures (hence in arterial input functions) and the limited coverage of the three MRR slices compared to the whole body projection of RR. Furthermore, at high local concentrations, signal becomes independent of T1 and is dominated by T2.

Adult↗

PubMed1982

Comparison of fungal mitochondrial introns reveals extensive homologies in RNA secondary structure.

The complete sequences of nine Saccharomyces cerevisiae mitochondrial introns, six of which carry long open reading frames, have already been published. We have recently determined the sequence of an intron in the large ribosomal mitochondrial RNA of Kluyveromyces thermotolerans (Jacquier et al., in preparation), which we found to be closely related to its S. cerevisiae counterpart. This latter result prompted us to undertake a systematic search for possible homologous elements in the other, available sequences with the help of an original computer program. A previously unsuspected wealth of evolutionarily conserved sequences and secondary structures was thus uncovered. Seven at least of the available sequences may be folded up into elaborate secondary structure models, the cores of which are nearly identical. These models result in bringing together the exon-intron junctions into relatively close spatial proximity and looping out either all or most of the sequences in open reading frame, when present. These results and their possible implications with respect to the mechanism of splicing are discussed in the light of available genetic and biochemical data.

Base Sequence↗

PubMed2004

High-throughput microarray detection of olfactory receptor gene expression in the mouse.

The large number of olfactory receptor genes necessitates high throughput methods to analyze their expression patterns. We have therefore designed a high-density oligonucleotide array containing all known mouse olfactory receptor (OR) and V1R vomeronasal receptor genes. This custom array detected a large number of receptor genes, demonstrating specific expression in the olfactory sensory epithelium for approximately 800 OR genes previously designated as ORs based solely on genomic sequences. The array also enabled us to monitor the spatial and temporal distribution of gene expression for the entire OR family. Interestingly, OR genes showing spatially segregated expression patterns were also segregated on the chromosomes. This correlation between genomic location and spatial expression provides unique insights about the regulation of this large family of genes.

Age Factors↗

PubMed1995

[Fast and ultra-fast magnetic resonance tomography. Basic principles, pulse sequences and special properties].

The aim of this article is the systematic treatment of fast and ultrafast magnetic resonance imaging (MRI) techniques. Based on the basic principles of signal generation and spatial encoding with magnetic field gradients the differences and important similarities of pulse sequences will be explained. We suggest to replace the conventional grouping of pulse sequences in gradient and spin-echo sequences through single and multi-echo sequences, since the latter is more precise and helpful. We illustrate how single-echo sequences such as "spin-echo", FLASH, FISP, PSIF, CISS and DESS can be derived from a single gradient echo and how multi-echo sequences such as turbo spin-echo RARE, HASTE and GRASE are based on echo-planar imaging. The different properties, advantages and disadvantages of the various sequences will be discussed and frequently used acronyms will be explained.

Echo-Planar Imaging↗

PubMed1998

3D multivoxel proton spectroscopy of human brain using a hybrid of 8th-order Hadamard encoding with 2D chemical shift imaging.

Multivoxel 3D localized proton spectroscopy using a hybrid of 1D 8th-order transverse Hadamard spectroscopic imaging (HSI) with 2D chemical shift imaging (CSI) is demonstrated in human brain. The spatially selective HSI pulse incorporates naturally into the PRESS sequence (TE = 135 ms), which then both excites an 8 x 8 x 6 cm parallelepiped volume of interest (VOI) and subdivides it into eight slices. The planes of these slices are further partitioned into 16 x 16 voxel arrays using 2D CSI to yield 8 x 8 x 8 voxels within the VOI. Simultaneous 3D coverage yields good voxel signal-to-noise (8, 12, and 22 for choline, creatine, and N-acetylaspartate, respectively) from these 0.75-ml voxels, in approximately 45 min. The high spatial isolation allows localization to within less than 1 cm from the skull without fat contamination.

Adult↗

PubMed2001

A high-resolution phantom for MRI.

Assessment of spatial resolution is an important step to test the performance of new sequence techniques-especially ultrafast techniques with dedicated k-space trajectories or interpolation algorithms. Measurement of the modulation transfer function (MTF) is a rather difficult procedure, but using suitable resolution phantoms allows a simple visual evaluation of spatial resolution. In contrast to commonly used test objects with a very restricted number of resolution patterns we developed a phantom containing resolution patterns from 0.1 to 1.5 mm in steps of 0.1 mm. One resolution pattern consists of five parallel Plexiglas strips with the distance of the strips being equal to their thickness. Together with a Plexiglas cuboid the resolution patterns are mounted on a Plexiglas plate on the bottom of the cylindrical phantom. An aqueous solution of manganese chloride is used to fill the phantom. High resolution cross sections (pixel size: 50 microm) through the resolution patterns were measured to confirm the correct dimensions of the phantom. To verify the appropriateness of the 0.1 and 0.2 mm stacks micro-CT images with a pixel size of 25 microm were acquired additionally for both patterns. Besides visual inspection evaluation of the profile function of signal intensity across the stacks demonstrates that the resolution patterns are sufficiently correct. T(1)-weighted SE sequences with slightly different pixel sizes as well as T(1)- and T(2*)- weighted gradient echo sequences were applied to demonstrate some possible applications of this phantom. In conclusion, the proposed phantom is well suited to assess the spatial resolution qualitatively (i.e., visually) and quantitatively over a wide range in steps of 0.1 mm.

Humans↗

PubMed2004

Free-breathing renal MR angiography with steady-state free-precession (SSFP) and slab-selective spin inversion: initial results.

BACKGROUND: The aim of our study was the investigation of a novel navigator-gated three-dimensional (3D) steady-state free-precession (SSFP) sequence for free-breathing renal magnetic resonance angiography (MRA) without contrast medium, and to examine the advantage of an additional inversion prepulse for improved contrast. METHODS: Eight healthy volunteers (mean age 29 years) and eight patients (mean age 53 years) were investigated on a 1.5 Tesla MR system (ACS-NT, Philips, Best, The Netherlands). Renal MRA was performed using three navigator-gated free-breathing cardiac-triggered 3D SSFP sequences [repetition time (TR) = 4.4 ms, echo time (TE) = 2.2 ms, flip angle 85 degrees, spatial resolution 1.25 x 1.25 x 4.0 mm(3), scanning time approximately 1 minute 30 seconds]. The same sequence was performed without magnetization preparation, with a non-slab selective and a slab-selective inversion prepulse. Signal-to-noise ratio (SNR), contrast-to-noise (CNR) vessel length, and subjective image quality were compared. RESULTS: Three-dimensional SSFP imaging combined with a slab-selective inversion prepulse enabled selective and high contrast visualization of the renal arteries, including the more distal branches. Standard SSFP imaging without magnetization preparation demonstrated overlay by veins and renal parenchyma. A non-slab-selective prepulse abolished vessel visualization. CNR in SSFP with slab-selective inversion was 43.6 versus 10.6 (SSFP without magnetization preparation) and 0.4 (SSFP with non-slab-selective inversion), P < 0.008. CONCLUSION: Navigator-gated free-breathing cardiac-triggered 3D SSFP imaging combined with a slab-selective inversion prepulse is a novel, fast renal MRA technique without the need for contrast media.

Adult↗

PubMed2003

Conspicuous histomorphological anomalies in the hippocampal formation of rats exposed prenatally to a complex sequenced magnetic field within the nanoTesla range.

The brains of adult rats, exposed prenatally to one of four intensities (between 10 nanoTesla and 1.2 microTesla) of either a frequency-modulated magnetic field or a complex sequenced field designed to affect brain development, were examined histologically. Although from each intensity some rats that had been exposed to the complex sequenced magnetic field showed minor anomalies, those exposed to intensities between 30 nT and 180 nT exhibited conspicuous anomalous organizations of cells within the hippocampal formation. In other studies, rats that had been exposed during their entire prenatal development to the complex sequenced field displayed significantly more activity in the open field and poorer spatial memory during maze learning. Photomicrographs are shown of one conspicuous morphological anomaly within the right hippocampus of an adult rat exposed prenatally to the complex sequenced magnetic field with intensities between .3 mG and .5 mG (30 nT to 50 nT). The results suggest that complex magnetic fields, whose temporal structures approach the time constants of normal biochemical processes, can permanently alter the development of the brain.

Animals↗

PubMed2001

Is aggregation of beta-amyloid peptides a mis-functioning of a current interaction process?

In a previous study, Hughes et al. [Proc. Natl. Acad. Sci. USA 93 (1996) 2065-2070] demonstrated that the amyloid peptide is able to interact with itself in a two-hybrid system and that interaction is specific. They further supported that the method could be used to define the sequences that might be important in nucleation-dependent aggregation. The sequence of the amyloid peptide can be split into four clusters, two hydrophilic (1-16 and 22-28) and two hydrophobic (17-21 and 29-42). We designed by molecular modeling and tested by the two-hybrid approach, series of mutations spread all over the sequence and changing the distribution of hydrophobicity and/or the spatial hindrance. In the two-hybrid assay, interaction of native Abeta is reproduced. Screening of mutations demonstrates that the C-domain (residues 29-40 (42)), the median domain (residues 17-22) and the N-domain (1-16) are all crucial for interaction. This demonstrates that almost all fragments of the amyloid peptide but a loop (residues 23-28) and the C-term amino acid are important for the native interaction. We support that the folded three-dimensional (3D) structure is the Abeta-Abeta interacting species, that the whole sequence is involved in that 3D fold which has a low secondary structure propensity and a high susceptibility to mutations and thus should have a low stability. The native fold of Abeta could be stabilized in Abeta-Abeta complexes which could in other circumstances facilitate the nucleation event of aggregation that leads to the formation of stable senile plaques.

Amino Acid Sequence↗

PubMed1997

Molecular divergence of lysozymes and alpha-lactalbumin.

The vast number of proteins that sustain the currently living organisms have been generated from a relatively small number of ancestral genes that has involved a variety of processes. Lysozyme is an ancient protein whose origin goes back an estimated 400 to 600 million years. This protein was originally a bacteriolytic defensive agent and has been adapted to serve a digestive function on at least two occasions, separated by nearly 40 million years. The origins of the related goose type and T4 phage lysozyme that are distinct from the more common C type are obscure. They share no discernable amino acid sequence identity and yet they possess common secondary and tertiary structures. Lysozyme C gene also gave rise, after gene duplication 300 to 400 million years ago, to a gene that currently codes for alpha-lactalbumin, a protein expressed only in the lactating mammary gland of all but a few species of mammals. It is required for the synthesis of lactose, the sugar secreted in milk. alpha-Lactalbumin shares only 40% identity in amino acid sequence with lysozyme C, but it has a closer spatial structure and gene organization. Although structurally similar, functionally they are quite distinct. Specific amino acid substitutions in alpha-lactalbumin account for the loss of the enzyme activity of lysozyme and the acquisition of the features necessary for its role in lactose synthesis. Evolutionary implications are as yet unclear but are being unraveled in many laboratories.

Amino Acid Sequence↗